Antithrombotic amides

ABSTRACT

This application relates to a compound of formula I (or a pharmaceutically acceptable salt thereof) as defined herein, pharmaceutical compositions thereof, and its use as an inhibitor of factor Xa, as well as a process for its preparation and intermediates therefor.

This application is a divisional of application Ser. No. 09/857,747,filed Jun. 8, 2001, now U.S. Pat. No. 6,610,704 B1, the national stageapplication of PCT/US99/29832, filed Dec. 15, 1999 and claims thebenefit of U.S. Provisional Application No. 60/113,778, filed Dec. 23,1998.

This invention relates to antithrombotic aromatic amides whichdemonstrate activity as inhibitors of factor Xa and, accordingly, whichare useful anticoagulants in mammals. In particular it relates toaromatic amides having high anticoagulant activity, and antithromboticactivity. Thus, this invention relates to new amides which areinhibitors of factor Xa, pharmaceutical compositions containing theamides as active ingredients, and the use of the amides asanticoagulants for prophylaxis and treatment of thromboembolic disorderssuch as venous thrombosis, pulmonary embolism, arterial thrombosis, inparticular myocardial ischemia, myocardial infarction and cerebralthrombosis, general hypercoagulable states and local hypercoagulablestates, such as following angioplasty and coronary bypass operations,and generalized tissue injury as it relates to the inflammatory process.In addition, the antithrombotic agents are useful as anticoagulants inin vitro applications.

The process of blood coagulation, thrombosis, is triggered by a complexproteolytic cascade leading to the formation of thrombin. Thrombinproteolytically removes activation peptides from the Aα-chains and theBβ-chains of fibrinogen, which is soluble in blood plasma, initiatinginsoluble fibrin formation. The formation of thrombin from prothrambinis catalyzed by factor Xa.

Anticoagulation currently is achieved by the administration of heparinsand coumarins. Parenteral pharmacological control of coagulation andthrombosis is based on inhibition of thrombin through the use ofheparins. Heparins act indirectly on thrombin by accelerating theinhibitory effect of endogenous antithrombin III (the main physiologicalinhibitor of thrombin). Because antithrombin III levels vary in plasmaand because clot-bound thrombin seems resistant to this indirectmechanism, heparins can be an ineffective treatment. Because coagulationassays are believed to be associated with efficacy and with safety,heparin levels must be monitored with coagulation assays (particularlythe activated partial thromboplastin time (APTT) assay). Coumarinsimpede the generation of thrombin by blocking the posttranslationalgamma-carboxylation in the synthesis of prothrombin and other proteinsof this type. Because of their mechanism of action, the effect ofcoumarins can only develop slowly, 6-24 hours after administration.Further, they are not selective anticoagulants. Coumarins also requiremonitoring with coagulation assays (particularly the prothrombin time(PT) assay).

Recently, interest has grown in small synthetic molecules whichdemonstrate potent direct inhibition of thrombin and factor Xa. See,Joseph P. Vacca (Annette M. Doherty Section Editor), Annual Reports inMedicinal Chemistry, (1998), 33, 81-90.

Although the heparins and coumarins are effective anticoagulants, therestill exists a need for anticoagulants which act selectively on factorXa or thrombin, and which, independent of antithrombin III, exertinhibitory action shortly after administration, preferably by an oralroute, and do not interfere with lysis of blood clots, as required tomaintain hemostasis.

The present invention is directed to the discovery that the amides ofthe present invention, as defined below, are potent inhibitors of factorXa which may have high bioavailability following oral administration.

According to the invention there is provided a compound of formula I

(or a pharmaceutically acceptable salt thereof) wherein:

A³, A⁴ A⁵ and A⁶, together with the two carbons to which they areattached, complete a substituted benzene in which A³ is CR³, A⁴ is CR⁴,A⁵ is CR⁵, and A⁶ is CR⁶;

wherein

R³ is hydrogen;

one of R⁴ and R⁵ is hydrogen, methyl, fluoro, chloro, R^(f)O₂C—, orR^(g)NH—;

the other of R⁴ and R⁵ is hydrogen; and

R⁶ is hydrogen;

in which R^(f) is hydrogen, (1-4C)alkyl or benzyl; R^(g) is hydrogen, orR^(h)SO₂—; and R^(h) is (1-4C)alkyl or dimethylamino; or

A³, A⁴, A⁵ and A⁶, together with the two carbons to which they areattached, complete a substituted heteroaromatic ring in which

(a) one of A³, A⁴, A⁵ and A⁶ is N, and each of the others is CR³, CR⁴,CR⁵ or CR⁶, respectively; or

(b) two non-adjacent residues of A³, A⁴, A⁵ and A⁶ are each N, and eachof the others is CR³, CR⁴, CR⁵ or CR⁶, respectively; wherein

each of R³, R⁴, R⁵ and R⁶ is independently hydrogen or methyl, or one ofR³, R⁴, R⁵ and R⁶ attached to a carbon which is not bonded to an N-atomis chloro and the others are hydrogen;

L¹ is —NH—CO—, —CO—NH— or —CH₂—NH— such that —L¹—Q¹ is —NH—CO—Q¹—CO—NH—Q¹ or —CH₂—NH—Q¹;

Q¹ is phenyl, 2-furanyl, 2-thienyl, 4-thiazolyl, 2-pyridyl, 2-naphthyl,1,2-dihydrobenzofuran-5-yl, 1,2-dihydrobenzofuran-6-yl,1,2-benzisoxazol-6-yl, 6-indolyl, 6-indolinyl, 6-indazolyl,5-benzimidazolyl or 5-benzotriazolyl in which the phenyl may bear one,two or three substituents at the 3-, 4- or 5-position(s) independentlyselected from halo, cyano, carbamoyl, aminomethyl, methyl, methoxy,difluoromethoxy, hydroxymethyl, formyl, vinyl, amino, hydroxy and3,4-methylenedioxy; and in addition the phenyl may bear a 2-chloro or2-fluoro substituent, the 2-furanyl or 2-thienyl may bear a chloro ormethyl substituent at the 5-position; the 4-thiazolyl may bear an aminosubstituent at the 2-position; the 2-pyridyl may bear an aminosubstituent at the 6-position; the 1,2-benzisoxazol-6-yl, 6-indolyl or6-indazolyl may bear a chloro or methyl substituent at the 3-position;or

—CO—Q¹ is cyclopentenylcarbonyl or cyclohexenylcarbonyl;

R² is —NH—CH₂—Q² in which Q² is Q²A or Q^(2B) wherein

Q^(2A) (showing the —CH₂— to which it is attached) is

 in which

R^(2A) is hydrogen, t-butyl, methylsulfonyl, —CHR^(y)R^(z),—CHR^(w)R^(x), or 4-pyridinyl (which is unsubstituted or bears asubstituent R^(v) at the 2- or 3-position) wherein

R^(v) is methyl, hydroxymethyl, {(1-2C)alkoxy}carbonyl; cyano,carbamoyl, thiocarbamoyl, or N-hydroxyamidino;

each of R^(w) and R^(x) independently is hydrogen or (1-3C)normal alkyl;or —CHR^(w)R^(x) is 2-indanyl or (showing the nitrogen to which it isattached) is

 in which T is a single bond or methylene and U is methylene, ethylene,oxy, —S(O)_(q)— (wherein q is 0, 1 or 2) or imino (which may bear amethyl substituent), or T is ethan-1,1-diyl and U is a single bond ormethylene;

R^(y) is hydrogen or methyl; and

R^(z) is isopropyl, t-butyl, (3-6C)cycloalkyl, phenyl (which isunsubstituted or bears one or more substituents independently selectedfrom halo, methyl, methoxy and hydroxy), 4-quinolinyl or heteroaryl(which heteroaryl is a 5-membered aromatic ring which includes one tofour heteroatoms selected from sulfur, oxygen and nitrogen or is a6-membered aromatic ring which includes one to three nitrogen atoms,wherein the heteroaryl is attached at carbon and may bear one or moremethyl substituents on carbon or nitrogen); and

Q^(2B) (showing the methylene to which it is attached) is

 in which R^(o) is hydrogen, halo, (1-6C)alkyl, hydroxy, (1-4C)alkoxy,benzyloxy or (1-4C)alkylthio; and R^(p) is 4-morpholinyl,1-hydroxyethyl, 1-hydroxy-1-methylethyl, 1-methoxy-1-methylethyl,4-piperidinyl, 4-pyridinyl, dimethylaminosulfonyl or —J—R^(q) in which Jis a single bond, methylene, carbonyl, oxy, —S(O)_(q)— (wherein q is 0,1 or 2), or —NR^(r)— (wherein R^(r) is hydrogen or methyl); and R^(q) is(1-6C)alkyl, phenyl, 3-pyridyl or 4-pyridyl.

As used herein, the expression a compound of formula I or the expressiona compound of the invention includes the compound and any conventionalprodrug thereof, as well as a pharmaceutically acceptable salt of saidcompound or prodrug.

A pharmaceutically acceptable salt of an antithrombotic agent of theinstant invention includes one which is an acid-addition salt made froma basic compound of formula I and an acid which provides apharmaceutically acceptable anion, as well as a salt which is made froman acidic compound of formula I and a base which provides apharmaceutically acceptable cation. Thus, a salt of a novel compound offormula I as provided herein made with an acid or base which affords apharmaceutically acceptable counterion provides a particular aspect ofthe invention. Examples of such acids and bases are providedhereinbelow.

As an additional aspect of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a novelcompound of formula I (or a pharmaceutically acceptable salt thereof) asprovided in any of the descriptions herein.

In addition, there is provided the use of a factor Xa inhibitingcompound of formula I (or prodrug or salt) as described herein as anactive ingredient in the manufacture of a medicament for use inproducing an anticoagulant or antithrombotic effect.

The present invention also provides a method of inhibiting coagulationin a mammal comprising administering to a mammal in need of treatment, acoagulation inhibiting dose of a factor Xa inhibiting compound offormula I having any of the definitions herein.

The present invention further provides a method of inhibiting factor Xacomprising administering to a mammal in need of treatment, a factor Xainhibiting dose of a factor Xa inhibiting compound of formula I havingany of the definitions herein.

Further, the present invention provides a method of treating athromboembolic disorder comprising administering to a mammal in need oftreatment, an effective done of a factor Xa inhibiting compound offormula I having any of the definitions herein.

In addition, there is provided the use of a factor Xa inhibitingcompound of formula I having any of the definitions herein for themanufacture of a medicament for treatment of a thromboembolic disorder.

As an additional feature of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a prodrug ofa factor Xa inhibiting compound of formula I (or of a pharmaceuticallyacceptable salt thereof) as provided in any of the descriptions herein.

In this specification, the following definitions are used, unlessotherwise described: Halo is fluoro, chloro, bromo or iodo. Alkyl,alkoxy, etc. denote both straight and branched groups; but reference toan individual radical such as “propyl” embraces only the straight chain(“normal”) radical, a branched chain isomer such as “isopropyl” beingspecifically denoted.

Particular values are listed below for radicals, substituents, andranges, for illustration only, and they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

For an alkyl group or the alkyl portion of an alkyl containing groupsuch as, for example alkoxy, a particular value for (1-2C)alkyl ismethyl or ethyl, and more particularly is methyl; for (1-3C)normal alkylis methyl, ethyl or propyl; for (1-4C)alkyl is methyl, ethyl, propyl,isopropyl, butyl, isobutyl, or t-butyl, and more particularly is methyl,isopropyl, butyl or t-butyl; for (1-6C)alkyl is methyl, ethyl, propyl,butyl, pentyl or hexyl, and more particularly is methyl, butyl, orhexyl. A particular value for (3-6C)cycloalkyl is cyclopropyl,cyclobutyl, cyclopenytyl or cyclohexyl. A particular value for halo isbromo or chloro, and more particularly is chloro.

A particular value for Q¹ is 4-chlorophenyl, 4-methoxy-phenyl,3-fluoro-4-methoxyphenyl, 5-chlorothiophen-2-yl, 2-pyridinyl or6-indolyl. A particular value for R² is 4-(4-morpholinyl)benzylamino,[1-(4-pyridinyl)piperin-4-yl-methyl]amino, or(1-isopropylpiperidin-4-ylmethyl)amino. When none of A³-A⁶ is N, aparticular set of values for R³-R⁶ is that each of R³-R⁶ is hydrogen;and another particular set of values for R³-R⁶ is that each of R³, R⁴and R⁶ is hydrogen and R⁵ is chloro. A further particular set of valuesis that A³ is N and each of A⁴-A⁶ is CH.

A particular value for —L¹—Q¹ is —CO—NH—Q¹.

Particular species are those listed below in the examples, and moreparticularly examples 8, 9, 11, 12, 14 and 15.

It will be appreciated that certain compounds of formula I (or salts orprodrugs, etc.) may exist in, and be isolated in, isomeric forms,including tautomeric forms, cis- or trans-isomers, as well as opticallyactive, racemic, or diastereomeric forms. It is to be understood thatthe present invention encompasses a compound of formula I in any of thetautomeric forms or as an a mixture thereof; or as a mixture ofdiastereomers, as well as in the form of an individual diastereomer, andthat the present invention encompasses a compound of formula I as amixture of enantiomers, as well as in the form of an individualenantiomer, any of which mixtures or form possesses inhibitoryproperties against factor Xa, it being well known in the art how toprepare or isolate particular forms and how to determine inhibitoryproperties against factor Xa by standard tests including those describedbelow.

In addition, a compound of formula I (or salt or prodrug, etc.) mayexhibit polymorphism or may form a solvate with water or an organicsolvent. The present invention also encompasses any such polymorphicform, any solvate or any mixture thereof.

A prodrug of a compound of formula I may be one formed in a conventionalmanner with a functional group of the compound, such as with an amino,hydroxy or carboxy group.

A compound of formula I may be prepared by processes which includeprocesses known in the chemical art for the production of structurallyanalogous compounds or by a novel process described herein. A processfor the preparation of a compound of formula I (or a pharmaceuticallyacceptable salt thereof) and novel intermediates for the manufacture ofa compound of formula I as defined above provide further features of theinvention and are illustrated by the following procedures in which themeanings of the generic radicals are as defined above, unless otherwisespecified. It will be recognized that it may be preferred or necessaryto prepare a compound of formula I in which a functional group isprotected using a conventional protecting group, then to remove theprotecting group to provide the compound of formula I.

Thus, there is provided a process for preparing a compound of formula I(or a pharmaceutically acceptable salt thereof) as provided in any ofthe above descriptions which is selected from any of those described inthe examples, including the following.

(A) For a compound of formula I in which —L¹—Q¹, is —NH—CO—Q¹, acylatingan amine of formula II,

using a corresponding acid of formula HO—CO—Q¹, or an activatedderivative thereof. Typical activated derivatives include the acidhalides, activated esters, including 4-nitrophenyl esters and thosederived from coupling reagents. Typical procedures include thatdescribed at Example 1-D.

(B) For a compound of formula I in which —L¹—Q¹ is —CO—NH—Q¹ and(preferably) at least one of A³ and A⁵ is N, substituting the groupY^(a) of a compound of formula III

in which Y^(a) is a conventional leaving group for nucleophilic aromaticsubstitution with an amine of formula NH₂—CH₂—Q². As used herein, aleaving group “Y^(a)” is a moiety which is displaced in an aromatic (orheteroaromatic) nucleophilic substitution reaction, for example a halogroup (such as fluoro or chloro), an alkoxy group (such as methoxy), asulfonate ester group (such as methylsulfonyloxy, p-toluyl-sulfonyloxyor trifluoromethylsulfonyloxy), or the reactive species derived fromtreating an alcohol with triphenyl-phospine, diethyl azodicarboxylateand triethyl amine (in a Mitsunobu reaction). The substitution may becarried out by heating a mixture of the reagents in a polar solvent, forexample in ethanol in a sealed tube as described at Example 8-B or indimethylformamide with cuprous bromide as described at example 11-B fora compound in which neither of A³ and A⁵ is N, but only A⁴ is N.

(C) For a compound of formula I in which —L¹—Q¹ is —CO—NH—Q¹, acylatingan amine of formula H₂N—Q¹, or a deprotonated derivative thereof, usingan acid of formula IV, or an activated derivative thereof.

Typical deprotonated derivatives of the amine H₂N—Q¹ include, forexample, that derived from treatment of the amine with anorganomagnesium reagent, for example, with allylmagnesium bromide ormethylmagnesium bromide. Typical activated derivatives include the acidhalides, activated esters, including 4-nitrophenyl esters and thosederived from coupling reagents. Preferably, the activated acid is ananhydride of formula IVb,

A typical procedure is that described at Example 12-B.

(D) Alkylating an amine of formula V

directly, using a compound of formula Y—CH₂—Q₂, as described at Example1-D, or (preferably) indirectly by reductive alkylation using analdehyde of formula Q²—CHO. In the reductive alkylation the intermediateimine of formula VI or acid addition salt thereof

(which provide a further aspect of the invention) may be formed in situ,and reduced directly, or may be isolated prior to reduction, for exampleas described at Example 14-E where the reduction is carried out usingborane trimethyl-amine complex in glacial acetic acid.

(E) For a compound of formula I in which —L¹—Q¹ is —CH₂—NH—Q¹, reducinga corresponding compound of formula I in which —L¹—Q¹ is —CO—NH—Q¹, forexample using lithium aluminum hydride in tetrahydrofuran as describedat Example 9.

(F) For a compound of formula I in which R^(2A) is methylsulfonyl,substituting the amino nitrogen of a corresponding compound of formula Iin which R^(2A) is hydrogen using an activated derivative ofmethanesulfonic acid, for example methanesulfonyl chloride in thepresence of added base.

(G) For a compound of formula I in which R^(2A) is —CHR^(y)R^(z) or—CHR^(w)R^(x), alkylating the amino nitrogen of a corresponding compoundof formula I in which R^(2A) is hydrogen using an alkylating agent offormula Y—CHR^(y)R^(z) or Y—CHR^(w)R^(x) or, preferably, reductivelyalkylating the amine using a compound of formula R^(y)—CO—R^(z) orR^(w)—CO—R^(x). The direct alkylation may be completed in a polarsolvent in the presence of a base. The reductive alkylation convenientlyis carried out, for example, using sodium cyanoborohydride inmethanol/acetic acid as described at Example 14-G or using sodiumtriacetoxyborohydride in an inert solvent such as 1,2-dichloroethanealong with an excess of the carbonyl compound and glacial acetic acid.

(H) For a compound of formula I in which R^(2A) is 4-pyridinyl (which isunsubstituted or bears a substituent R^(v) at the 2- or 3-position),substituting the amino nitrogen of a corresponding compound of formula Iin which R^(2A) is hydrogen using a corresponding pyridine reagentbearing a leaving group Y at the 4-position, for example with a4-chloropyridine in ethanol.

(I) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is alkoxycarbonyl, esterifying a corresponding compound of formulaI in which R^(v) is carboxy.

(J) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is hydroxymethyl, reducing the ester of a corresponding compoundof formula I in which R^(v) is alkoxycarbonyl.

(K) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is carbamoyl, amidating the ester of a corresponding compound offormula I in which R^(v) is alkoxycarbonyl.

(L) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is thiocarbamoyl, adding H₂S to the nitrile of a correspondingcompound of formula I in which R^(v) is cyano.

(M) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is N-hydroxyamidino, adding H₂NOH to the nitrile of acorresponding compound of formula I in which R^(v) is cyano. Theaddition may be direct or indirect, such as via an imidate ester or bytreating a compound in which R^(v) is thiocarbamoyl with methyl iodideto form a thioimidate ester, then treatment with hydroxylamine.

(N) For a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is carboxy, decomposing the ester of a corresponding compound offormula I in which R^(v) is alkoxycarbonyl.

(O) For a compound of formula I in which —NR^(s)R^(t) is other thanamino, alkylating a corresponding compound of formula I in which—NR^(s)R^(t) is amino using a conventional method. When R^(s) and R^(t)together are trimethylene or tetramethylene, a difunctional alkylatingagent, such as 1,3-dibromopropane or 1,4-dibromobutane is preferred.

(P) For a compound of formula I which bears —NR^(s)R^(t), reductivelyalkylating H—NR^(s)R^(t) using a corresponding compound but in which thecarbon to bear the —NR^(s)R^(t) group bears an oxo group, for example,using a procedure similar to one of procedure (G) above.

(Q) For a compound of formula I in which R^(p) is1-hydroxy-1-methylethyl, adding a methyl group to the carbonyl group ofa corresponding compound of formula I in which R^(p) is acetyl using anorganometallic reagent such as, for example, methylmagnesium bromide.

(R) For a compound of formula I in which R^(p) is1-methoxy-1-methylethyl, treating a corresponding compound of formula Iin which R^(p) is 1-hydroxy-1-methylethyl with methanol and an acidcatalyst.

(S) For a compound of formula I in which R⁴ or R⁵ is amino, reducing thenitro group of a compound corresponding to a compound of formula I butin which R⁴ or R⁵ is nitro.

(T) For a compound of formula I in which R⁴ or R⁵ is R^(g)NH— and R^(g)is R^(h)SO₂—, substituting the amino group of a corresponding compoundof formula I in which R⁴ or R⁵ is amino using an activated derivative ofthe sulfonic acid R^(h)SO₂—OH.

Whereafter, for any of the above procedures, when a functional group isprotected using a protecting group, removing the protecting group.

Whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of formula I is required, it is obtainedby reacting the basic for of a basic compound of formula I with an acidaffording a physiologically acceptable counterion or the acidic form ofan acidic compound of formula I with a base affording a physiologicallyacceptable counterion or by any other conventional procedure.

A novel intermediate or starting material compound such as, for example,a novel compound of formula II, III, IV or VI, etc., provides a furtheraspect of the invention. The various starting material may be made byprocesses which include processes known in the chemical art for theproduction of structurally analogous compounds or by a novel processdescribed herein or one analogous thereto.

As mentioned above, a compound corresponding to a compound of formula Ibut in which a functional group is protected may serve as anintermediate for a compound of formula I. Accordingly, such a protectedintermediate for a novel compound of formula I provides a further aspectof the invention. Thus, as one particular aspect of the invention, thereis provided a compound corresponding to a novel compound of formula I asdefined above in which R⁴ is hydroxy, but in which the correspondingsubstituent is —OP^(p) in place of hydroxy, wherein P^(p) is a phenolprotecting group other than (1-4C)alkyl or benzyl. Phenol protectinggroups are well known in the art, for example as described in T. W.Greene and P. G. M. Wuts, “Protecting Groups in Organic Synthesis”(1991). Further, P^(p) may denote a functionalized resin, for example asdisclosed in H. V. Meyers, et al., Molecular Diversity, (1995), 1,13-20.

As mentioned above, the invention includes a pharmaceutically acceptablesalt of the factor Xa inhibiting compound defined by the above formulaI. A basic compound of this invention possesses one or more functionalgroups sufficiently basic to react with any of a number of inorganic andorganic acids affording a physiologically acceptable counterion to forma pharmaceutically acceptable salt. Acids commonly employed to formpharmaceutically acceptable acid addition salts are inorganic acids suchas hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,bexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferredpharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid, hydrobromic acid andsulfuric acid.

For a compound of formula I which bears an acidic moiety, such as acarboxy group, a pharmaceutically acceptable salt may be made with abase which affords a pharmaceutically acceptable cation, which includesalkali metal salts (especially sodium and potassium), alkaline earthmetal salts (especially calcium and magnesium), aluminum salts andammonium salts, as well as salts made from physiologically acceptableorganic bases such as triethylamine, morpholine, piperidine andtriethanolamine.

If not commercially available, a necessary starting material for thepreparation of a compound of formula I may be prepared by a procedurewhich is selected from standard techniques of organic chemistry,including aromatic and heteroaromatic substitution and transformation,from techniques which are analogous to the syntheses of known,structurally similar compounds, and techniques which are analogous tothe above described procedures or procedures described in the Examples.It will be clear to one skilled in the art that a variety of sequencesis available for the preparation of the starting materials. Startingmaterials which are novel provide another aspect of the invention.

Selective methods of substitution, protection and deprotection are wellknown in the art for preparation of a compound such as one of formulaII, III, IV or VI discussed above.

Generally, a basic compound of the invention is isolated best in theform of an acid addition salt. A salt of a compound of formula I formedwith an acid such as one of those -mentioned above is useful as apharmaceutically acceptable salt for administration of theantithrombotic agent and for preparation of a formulation of the agent.Other acid addition salts may be prepared and used in the isolation andpurification of the compounds.

As noted above, the optically active isomers and diastereomers of thecompounds of formula I are also considered part of this invention. Suchoptically active isomers may be prepared from their respective opticallyactive precursors by the procedures described above, or by resolving theracemic mixtures. This resolution can be carried out by derivatizationwith a chiral reagent followed by chromatography or by repeatedcrystallization. Removal of the chiral auxiliary by standard methodsaffords substantially optically pure isomers of the compounds of thepresent invention or their precursors. Further details regardingresolutions can be obtained in Jacques, et al., Enantiomers, Racemates,and Resolutions, John Wiley & Sons, 1981.

The compounds of the invention are believed to selectively inhibitfactor Xa over other proteinases and nonenzyme proteins involved inblood coagulation without appreciable interference with the body'snatural clot lysing ability (the compounds have a low inhibitory effecton fibrinolysis). Further, such selectivity is believed to permit usewith thrombolytic agents without substantial interference withthrombolysis and fibrinolysis.

The invention in one of its aspects provides a method of inhibitingfactor Xa in mammals comprising administering to a mammal in need oftreatment an effective (factor Xa inhibiting) dose of a compound offormula I.

In another of its aspects, the invention provides a method of treating athromboembolic disorder comprising administering to a mammal in need oftreatment an effective (thromboembolic disorder therapeutic and/orprophylactic amount) dose of a compound of formula I.

The invention in another of its aspects provides a method of inhibitingcoagulation in a mammal comprising administering to a mammal in need oftreatment an effective (coagulation inhibiting) dose of a compound offormula I.

The factor Xa inhibition, coagulation inhibition and thromboembolicdisorder treatment contemplated by the present method includes bothmedical therapeutic and/or prophylactic treatment as appropriate.

In a further embodiment, the invention relates to treatment, in a humanor animal, of a condition where inhibition of factor Xa is required. Thecompounds of the invention are expected to be useful in mammals,including man, in treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues. Disorders in which thecompounds have a potential utility are in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. Disorders inwhich the compounds have a potential utility, in treatment and/orprophylaxis, include venous thrombosis and pulmonary embolism, arterialthrombosis, such as in myocardial ischemia, myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compounds have expected utility in thetreatment or prophylaxis of atherosclerotic disorders (diseases) such ascoronary arterial disease, cerebral arterial disease and peripheralarterial disease. Further, the compounds are expected to be usefultogether with thrombolytics in myocardial infarction. Further, thecompounds have expected utility in prophylaxis for reocclusion afterthrombolysis, percutaneous transluminal angioplasty (PTCA) and coronarybypass operations. Further, the compounds have expected utility inprevention of rethrombosis after microsurgery. Further, the compoundsare expected to be useful in anticoagulant treatment in connection withartificial organs, including joint replacement, and cardiac valves.Further, the compounds have expected utility in anticoagulant treatmentin hemodialysis and disseminated intravascular coagulation. A furtherexpected utility is in rinsing of catheters and mechanical devices usedin patients in vivo, and as an anticoagulant for preservation of blood,plasma and other blood products in vitro. Still further, the compoundshave expected utility in other diseases where blood coagulation could bea fundamental contributing process or a source of secondary pathology,such as cancer, including metastasis, inflammatory diseases, includingarthritis, and diabetes. The anti-coagulant compound is administeredorally or parenterally, e.g. by intravenous infusion (iv), intramuscularinjection (im) or subcutaneously (sc).

The specific dose of a compound administered according to this inventionto obtain therapeutic and/or prophylactic effects will, of course, bedetermined by the particular circumstances surrounding the case,including, for example, the compound administered, the rate ofadministration, the route of administration, and the condition beingtreated.

A typical daily dose for each of the above utilities is between about0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate. In critical caresituations a compound of the invention is administered by iv infusion ata rate between about 0.1 mg/kg/h and about 20 mg/kg/h and preferablybetween about 0.1 mg/kg/h and about 5 mg/kg/h.

The method of this invention also is practiced in conjunction with aclot lysing agent e.g. tissue plasminogen activator (t-PA), modifiedt-PA, streptokinase or urokinase. In cases when clot formation hasoccurred and an artery or vein is blocked, either partially or totally,a clot lysing agent is usually employed. A compound of the invention canbe administered prior to or along with the lysing agent or subsequent toits use, and preferably further is administered along with aspirin toprevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction with aplatelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibitsplatelet aggregation. A compound of the invention can be administeredprior to or along with the IIb/IIIa antagonist or subsequent to its useto prevent the occurrence or reoccurrence of clot formation.

The method of this invention is also practiced in conjunction withaspirin. A compound of the invention can be administered prior to oralong with aspirin or subsequent to its use to prevent the occurrence orreoccurrence of clot formation. As stated above, preferably a compoundof the present invention is administered in conjunction with a clotlysing agent and aspirin.

This invention also provides a pharmaceutical composition for use in theabove described therapeutic method. A pharmaceutical composition of theinvention comprises an effective factor Xa inhibiting amount of acompound of formula I in association with a pharmaceutically acceptablecarrier, excipient or diluent.

The active ingredient in such formulations comprises from 0.1 percent to99.9 percent by weight of the formulation. By “pharmaceuticallyacceptable” it is meant the carrier, diluent or excipient must becompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

For oral administration the antithrombotic compound is formulated ingelatin capsules or tablets which may contain excipients such asbinders, lubricants, disintegration agents and the like. For parenteraladministration the antithrombotic is formulated in a pharmaceuticallyacceptable diluent e.g. physiological saline (0.9 percent), 5 percentdextrose, Ringer's solution and the like.

The compound of the present invention can be formulated in unit dosageformulations comprising a dose between about 0.1 mg and about 1000 mg.Preferably the compound is in the form of a pharmaceutically acceptablesalt such as for example the sulfate salt, acetate salt or a phosphatesalt. An example of a unit dosage formulation comprises 5 mg of acompound of the present invention as a pharmaceutically, acceptable saltin a 10 mL sterile glass ampoule. Another example of a unit dosageformulation comprises about 10 mg of a compound of the present inventionas a pharmaceutically acceptable salt in 20 mL of isotonic salinecontained in a sterile ampoule.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. The compounds of the present invention are preferablyformulated prior to administration.

The present pharmaceutical compositions are prepared by known proceduresusing well known and readily available ingredients. The compositions ofthis invention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. “Activeingredient,” of course, means a compound according to formula I or apharmaceutically acceptable salt or solvate thereof.

Quantity (mg/capsule) Formulation 1: Hard gelatin capsules are preparedusing the following ingredients: Active ingredient 250 Starch, dried 200Magnesium stearate  10 Total 460 mg Formulation 2: A tablet is preparedusing the ingredients below: Active ingredient 250 Cellulose,microcrystalline 400 Silicon, dioxide, fumed  10 Stearic acid  5 Total665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3: An aerosol solution is preared containing the followingcomponents: Weight Active ingredient 0.25 Ethanol 29.75 Propellant 22(Chlorodifluoromethane) 70.00 Total 100.00

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to −30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulartion 4: Tablets, each containing 60 mg of active ingredient, aremade as follows: Active ingredient 60 mg Starch 45 mg Microcrystallinecellulose 35 mg Polyvinylpyrrolidone (as 10% solution in 4 mg water)Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mgTotal 150 mg

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinylpyrrolidone is mixed with the resultant powder, and the mixturethen is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C. and passed through a No. 18 mesh U.S.Sieve. The sodium carboxymethyl starch, magnesium stearate and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

Formulation 5: Capsules, each containing 80 mg of active ingredient, aremade as follows: Active ingredient  80 mg Starch  59 mg Microcrystallinecellulose  59 mg Magnesium stearate  2 mg Total 200 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6: Suppositories, each containing 225 mg of activeingredient, are made as follows: Active ingredient   225 mg Saturatedfatty acid glycerides 2,000 mg Total 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7: Suspensions, each containing 50 mg of active ingredientper 5 mL dose, are made as follows: Active ingredient 50 mg Sodiumcarboxymethyl cellulose 50 mg Syrup 1.25 mL Benzoic acid solution 0.10mL Flavor q.v. Color q.v. Purified water to total 5 mL

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8: An intravenous formulation may be prepared as follows:Active ingredient 100 mg Isotonic saline 1,000 mL

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 mL per minute.

The ability of a compound of the present invention to be an effectiveand orally active factor Xa inhibitor may be evaluated in one or more ofthe following assays or in other standard assays known to those in theart.

The inhibition by a compound of the inhibition of a serine protease ofthe human blood coagulation system or of the fibrinolytic system, aswell as of trypsin, is determined in vitro for the particular enzyme bymeasuring its inhibitor binding affinity in an assay in which the enzymehydrolyzes a particular chromogenic substrate, for example as describedin Smith, G. F.; Gifford-Moore, D.; Craft, T. J.; Chirgadze, N.;Ruterbories, K. J.; Lindstrom, T. D.; Satterwhite, J. H. Efegatran: ANew Cardiovascular Anticoagulant. New Anticoagulants for theCardiovascular Patient; Pifarre, R., Ed.; Hanley & Belfus, Inc.:Philadelphia, 1997; pp. 265-300. The inhibitor binding affinity ismeasured as apparent association constant Kass which is the hypotheticalequilibrium constant for the reaction between enzyme and the testinhibitor compound (I). Enzyme + I ⇄ Enzyme − I${Kass} = \frac{\left\lbrack {{Enzyme} - I} \right\rbrack}{\left\lbrack {({Enzyme}) \times (I)} \right\rbrack}$

Conveniently, enzyme inhibition kinetics are performed in 96-wellpolystyrene plates and reaction rates are determined from the rate ofhydrolysis of appropriate p-nitroanilide substrates at 405 nm using aThermomax plate reader from Molecular Devices (San Francisco, Calif.).The same protocol is followed for all enzymes studied: 50 μL buffer(0.03 M Tris, 0.15 M NaCl pH 7) in each well, followed by 25 μL ofinhibitor solution (in 100% methanol, or in 50% v:v aqueous methanol)and 25 μL enzyme solution; within two minutes, 150 μL aqueous solutionof chromogenic substrate (0.25 mg/mL) is added to start the enzymaticreaction. The rates of chromogenic substrate hydrolysis reactionsprovide a linear relationship with the enzymes studied such that freeenzyme can be quantitated in reaction mixtures. Data is analyzeddirectly as rates by the Softmax program to produce [free enzyme]calculations for tight-binding Kass determinations. For apparent Kassdeterminations, 1.34 nM human factor Xa is used to hydrolyze 0.18 mMBzIle-Glu-Gly-Arg-pNA; 5.9 nm human thrombin or 1.4 nM bovine trypsin isused to hydrolyze 0.2 mM BzPhe-Val-Arg-pNA; 3.4 nM human plasmin is usedwith 0.5 mM ED-Val-Leu-Lys-pNA; 1.2 nM human nt-PA is used with 0.81 mMHD-IIe-Pro-Arg-pNA; and 0.37 nM urokinase is used with 0.30 mMpyro-gfsGlu-Gly-Arg-pNA.

Kass is calculated for a range of concentrations of test compounds andthe mean value reported in units of liter per mole. In general, a factorXa inhibiting compound of formula I of the instant invention, asexemplified herein, exhibits a Kass of 0.1 to 0.5×10⁶ L/mole or muchgreater.

The factor Xa inhibitor preferably should spare fibrinolysis induced byurokinase, tissue plasminogen activator (t-PA) and streptokinase. Thiswould be important to the therapeutic use of such an agent as an adjunctto streptokinase, tp-PA or urokinase thrombolytic therapy and to the useof such an agent as an endogenous fibrinolysis-sparing (with respect tot-PA and urokinase) antithrombotic agent. In addition to the lack ofinterference with the amidase activity of the fibrinolytic proteases,such fibrinolytic system sparing can be studied by the use of humanplasma clots and their lysis by the respective fibrinolytic plasminogenactivators.

Materials

Dog plasma is obtained from conscious mixed-breed hounds (either sexButler Farms, Clyde, N.Y., U.S.A.) by venipuncture into 3.8 percentcitrate. Fibrinogen is prepared from fresh dog plasma and humanfibrinogen is prepared from in-date ACD human blood at the fraction I-2according to previous procedures and specification. Smith, Biochem. J.,185, 1-11 (1980; and Smith, et al., Biochemistry, 11, 2958-2967, (1972).Human fibrinogen (98 percent pure/plasmin free) is from AmericanDiagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen I-2preparations is performed as previously reported. Smith, et al.,Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased from LeoPharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase ispurchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.

Methods—Effects on Lysis of Human Plasma Clots by t-PA

Human plasma clots are formed in micro test tubes by adding 50 μLthrombin (73 NIH unit/mL) to 100 μL human plasma which contains 0.0229μCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlayingthe clots with 50 μL of urokinase or streptokinase (50, 100, or 1000unit/mL) and incubating for 20 hours at room temperature. Afterincubation the tubes are centrifuged in a Beckman Microfuge. 25 μL ofsupernate is added into 1.0 mL volume of 0.03 M tris/0.15 M NaCl bufferfor gunun counting. Counting controls 100 percent lysis are obtained byomitting thrombin (and substituting buffer). The factor Xa inhibitorsare evaluated for possible interference with fibrinolysis by includingthe compounds in the overlay solutions at 1, 5, and 10 μg/mLconcentrations. Rough approximations of IC₅₀ values are estimated bylinear extrapolations from data points to a value which would represent50 percent of lysis for that particular concentration of fibrinolyticagent.

Anticoagulant Activity

Materials

Dog plasma and rat plasma are obtained from conscious mixed-breed hounds(either sex, Butler Farms, Clyde, N.Y., U.S.A.) or from anesthetizedmale Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis,Ind., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen isprepared from in-date ACD human blood as the fraction I-2 according toprevious procedures and specifications. Smith, Biochem. J., 185, 1-11(1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972). Ruianfibrinogen is also purchased as 98 percent pure/plasmin free fromAmerican Diagnostica, Greenwich, Conn. Coagulation reagents Actin,Thromboplastin, Innovin and Human plasma are from Baxter HealthcareCorp., Dade Division, Miami, Fla. Bovine thrombin from Parke-Davis(Detroit, Mich.) is used for coagulation assays in plasma.

Methods

Anticoagulation Determinations

Coagulation assay procedures are as previously described. Smith, et al.,Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulationinstrument (American LABor, Inc.) is used for all coagulation assaymeasurements. The prothrombin time (PT) is measured by adding 0.05 mLsaline and 0.05 mL Thromboplastin-C reagent or recombinant human tissuefactor reagent (Innovin) to 0.05 mL test plasma. The activated partialthromboplastin time (APTT) is measured by incubation of 0.05 mL testplasma with 0.05 mL Actin reagent for 120 seconds followed by 0.05 mLCaCl₂ (0.02 M). The thrombin time (TT) is measured by adding 0.05 mLsaline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mL test plasma.The compounds of formula I are added to human or animal plasma over awide range of concentrations to determine prolongation effects on theAPTT, PT, and TT assays. Linear extrapolations are performed to estimatethe concentrations required to double the clotting time for each assay.

Animals

Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc.,Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) andketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37°C.). The jugular vein(s) in cannulated to allow for infusions.

Arterio-Venous Shunt Model

The left jugular vein and right carotid artery are cannulated with 20 cmlengths of polyethylene PE 60 tubing. A 6 cm center section of largertubing (PE 190) with a cotton thread (5 cm) in the lumen, is frictionfitted between the longer sections to complete the arterio-venous shuntcircuit. Blood is circulated through the shunt for 15 min before thethread is carefully removed and weighed. The weight of a wet thread issubtracted from the total weight of the thread and thrombus (see J. R.Smith, Br J Pharmacol, 77:29, 1982).

PeCl₃ Model of Arterial Injury

The carotid arteries are isolated via a midline ventral cervicalincision. A thermocouple is placed under each artery and vesseltemperature is recorded continuously on a strip chart recorder. A cuffof tubing (0.058 ID×0.077 OD×4 mm, Baxter Med. Grade Silicone), cutlongitudinally, is placed around each carotid directly above thethermocouple. FeCl₃ hexahydrate is dissolved in water and theconcentration (20 percent) is expressed in terms of the actual weight ofFeCl₃ only. To injure the artery and induce thrombosis, 2.85 μL ispipetted into the cuff to bathe the artery above the thermocouple probe.Arterial occlusion is indicated by a rapid drop in temperature. The timeto occlusion is reported in minutes and represents the elapsed timebetween application of FeCl₃ and the rapid drop in vessel temperature(see K. D. Kurz, Thromb. Res., 60:269, 1990).

Coagulation Parameters

Plasma thrombin time (TT) and activated partial thromboplastin time(APTT) are measured with a fibrometer. Blood is sampled from a jugularcatheter and collected in syringe containing sodium citrate (3.8percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 mL) ismixed with saline (0.1 mL) and bovine thrombin (0.1 mL, 30 U/mL in TRISbuffer; Parke Davis) at 37° C. For APTT, plasma (0.1 mL) and APTTsolution (0.1 mL, Organon Teknika) are incubated for 5 minutes (37° C.)and CaCl₂ (0.1 mL, 0.025 M) is added to start coagulation. Assays aredone in duplicate and averaged.

Index of Bioavailability

Bioavailability studies may be conducted as follows. Compounds areadministered as aqueous solutions to male Fisher rats, intravenously(iv) at 5 mg/kg via tail vein injection and orally (po) to fastedanimals at 20 mg/kg by gavage. Serial blood samples are obtained at 5,30, 120, and 240 minutes postdose following intravenous administrationand at 1, 2, 4, and 6 hours after oral dosing. Plasma is analyzed fordrug concentration using an HPLC procedure involving CB Bond Elute(Varion) cartridges for sample preparation and a methanol/30 nM ammoniumacetate buffer (pH 4) gradient optimized for each compound. % Oralbioavailability is calculated by the following equation:${\% \quad {Oral}\quad {bioavailability}} = {\frac{{AUC}\quad {po}}{{AUC}\quad {iv}} \times \frac{{Dose}\quad {iv}}{{Dose}\quad {po}} \times 100}$

where AUC is area under the curve calculated from the plasma level ofcompound over the time course of the experiment following oral (AUC po)and intravenous (AUC iv) dosing.

Compounds

Compound solutions are prepared fresh daily in normal saline and areinjected as a bolus or are infused starting 15 minutes before andcontinuing throughout the experimental perturbation which is 15 minutesin the arteriovenous shunt model and 60 minutes in the FeCl₃ model ofarterial injury and in the spontaneous thrombolysis model. Bolusinjection volume is 1 mL/kg for i.v., and 5 mL/kg for p.o., and infusionvolume is 3 mL/hr.

Statistics

Results are expressed as means+/−SEM. One-way analysis of variance isused to detect statistically significant differences and then Dunnett'stest is applied to determine which means are different. Significancelevel for rejection of the null hypothesis of equal means is P<0.05.

Animals

Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, NorthRose, N.Y. 14516) are fasted overnight and fed Purina certifiedPrescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes afterdosing. Water is available ad libitum. The room temperature ismaintained between 66-74° F.; 45-50 percent relative humidity; andlighted from 0600-1800 hours.

Pharmacokinetic Model

Test compound is formulated immediately prior to dosing by dissolving insterile 0.9 percent saline to a 5 mg/mL preparation. Dogs are given asingle 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5mL) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and6 hours after dosing. Samples are collected in citrated Vacutainer tubesand kept on ice prior to reduction to plasma by centrifugation. Plasmasamples are analyzed by HPLC MS. Plasma concentration of test compoundis recorded and used to calculate the pharmacokinetic parameters:elimination rate constant, Ke; total clearance, Clt; volume ofdistribution, V_(D); time of maximum plasma test compound concentration,Tmax; maximum concentration of test compound of Tmax, Cmax; plasmahalf-life, t0.5; and area under the curve, A.U.C.; fraction of testcompound absorbed, F.

Canine Model of Coronary Artery Thrombosis

Surgical preparation and instrumentation of the dogs are as described inJackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds(aged 6-7 months, either sex, Butler Farms, Clyde, N.Y., U.S.A.) areanesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.),intubated, and ventilated with room air. Tidal volume and respiratoryrates are adjusted to maintain blood PO₂, PCO₂, and pH within normallimits. Subdermal needle electrodes are inserted for the recording of alead II ECG.

The left jugular vein and common carotid artery are isolated through aleft mediolateral neck incision. Arterial blood pressure (ASP) ismeasured continuously with a precalibrated Millar transducer (model(MPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into thecarotid artery. The jugular vein is cannulated for blood sampling duringthe experiment. In addition, the femoral veins of both hindlegs arecannulated for administration of test compound.

A left thoracotomy is performed at the fifth intercostal space, and theheart is suspended in a pericardial cradle. A 1- to 2-cm segment of theleft circumflex coronary artery (LCX) is isolated proximal to the firstmajor diagonal ventricular branch. A 26-gauge needle-tipped wire anodalelectrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm longis inserted into the LCX and placed in contact with the intimal surfaceof the artery (confirmed at the end of the experiment). The stimulatingcircuit is completed by placing the cathode in a subcutaneous (s.c.)site. An adjustable plastic occluder is placed around the LCX, over theregion of the electrode. A precalibrated electromagnetic flow probe(Carolina Medical Electronics, King, N.C., U.S.A.) is placed around theLCX proximal to the anode for measurement of coronary blood flow (CBF).The occluder is adjusted to produce a 40-50 percent inhibition of thehyperemic blood flow response observed after 10-s mechanical occlusionof the LCX. All hemodynamic and ECG measurements are recorded andanalyzed with a data acquisition system (model M3000, ModularInstruments, Malvern, Pa. U.S.A.).

Thrombus Formation and Compound Administration Regimens

Electrolytic injury of the intima of the LCX is produced by applying100-μA direct current (DC) to the anode. The current is maintained for60 min and then discontinued whether the vessel has occluded or not.Thrombus formation proceeds spontaneously until the LCX is totallyoccluded (determined as zero CBF and an increase in the S-T segment).Compound administration is started after the occluding thrombus isallowed to age for 1 hour. A 2-hour infusion of the compounds of thepresent invention at doses of 0.5 and 1 mg/kg/hour is begunsimultaneously with an infusion of thrombolytic agent (e.g. tissueplasminogen activator, streptokinase, APSAC). Reperfusion is followedfor 3 hour after administration of test compound. Reocclusion ofcoronary arteries after successful thrombolysis is defined as zero CBFwhich persisted for at least 30 minutes.

Hematology and Template Bleeding Time Determinations

Whole blood cell counts, hemoglobin, and hematocrit values aredetermined on a 40-AL sample of citrated (3.8 percent) blood (1 partcitrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900,Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleedingtimes are determined with a Simplate II bleeding time device (OrganonTeknika Durham, N.C., U.S.A.). The device is used to make 2 horizontalincisions in the gingiva of either the upper or lower left jaw of thedog. Each incision is 3 mm wide×2 mm deep. The incisions are made, and astopwatch is used to determine how long bleeding occurs. A cotton swabis used to soak up the blood as it oozes from the incision. Templatebleeding time is the time from incision to stoppage of bleeding.Bleeding times are taken just before administration of test compound (0min), 60 min into infusion, at conclusion of administration of the testcompound (120 min), and at the end of the experiment.

All data are analyzed by one-way analysis of variance (ANOVA) followedby Student-Neuman-Kuels post hoc t test to determine the level ofsignificance. Repeated-measures ANOVA are used to determine significantdifferences between time points during the experiments. Values aredetermined to be statistically different at least at the level ofp<0.05. All values are mean±SEM. All studies are conducted in accordancewith the guiding principles of the American Physiological Society.Further details regarding the procedures are described in Jackson, etal., J. Cardiovasc. Pharmacol., (1993), 21, 587-599.

The following Examples are provided to further describe the inventionand are not to be construed as limitations thereof.

The abbreviations, symbols and terms used in the examples have thefollowing meanings.

Ac=acetyl

Boc=t-butyloxycarbonyl

Calcd=calculated

conc=concentrated

DMF=dimethylformamide

DMSO=dimethylsulfoxide

Et=ethyl

EtOAc=ethyl acetate

EtOH=ethanol

FTIR=Fourier transform IR

HPLC=High Performance Liquid Chromatography

HRMS=high resolution mass spectrum

i-PrOH=isopropanol

IR=Infrared Spectrum

LAH=lithium aluminum hydride

LC-MS=liquid chromatography—mass spectrum (using HPLC)

Me=methyl

MeOH=methanol

MS-ES (or ES-MS)=electrospray mass spectrum

MS-FAB (or FAB-MS) fast atom bombardment mass spectrum

MS-FIA (or FIA-MS)=flow injection analysis mass spectrum

MS-FD (or FD-MS)=field desorption mass spectrum

MS-IS (or IS-MS)=ion spray mass spectrum

MNR=Nuclear Magnetic Resonance

Ph=phenyl

i-Pr=isopropyl

RPHPLC=Reversed Phase High Performance Liquid Chromatography

RT (or R_(t))=retention time

satd=saturated

SiO₂=silica gel

SCX=strong cation exchange (resin)

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TLC=thin layer chromatography

Unless otherwise stated, pH adjustments and work up are with aqueousacid or base solutions. ¹H-NMR indicates a satisfactory NMR spectrum wasobtained for the compound described. IR (or FTIR) indicates asatisfactory infra red spectrum was obtained for the compound described.

EXAMPLE 1 Preparation ofN¹-(4-Methoxybenzoyl)-N²-[4-(4-morpholinyl)-benzyl]-(1,2-benzenediamine

A. 4-(4-morpholinyl)benzonitrile

A solution of 4-fluorobenzonitrile (1.00 g, 8.26 mmol) and morpholine(0.77 mL, 9.08 mmol) in dimethyl sulfoxide (40 mL) was treated with 37%KF on alumina, and the mixture was heated at 150° C. for 5 h. Aftercooling, the mixture was diluted with EtOAc and filtered throughdiatomaceous earth. The filtrate was washed with water (3×), brine (1×),and then dried with magnesium sulfate. The extract was concentrated andthe residue was purified by chromatography (SiO₂, 20 to 30% EtOAc inhexanes) yielding 930 mg (60%) of the title compound.

¹NMR

B. 4-(4-morpholinyl)benzoic acid

A solution of 4-(4-morpholinyl)benzonitrile (930 mg, 5.00 mmol) in 1:1dioxane:water (20 mL) was treated with potassium hydroxide (1.12 g, 20mmol). The mixture was heated at reflux for 96 h, concentrated, and theresidue was dissolved in water. Upon acidification (pH˜2-3), a whiteprecipitate resulted which was collected by filtration yielding 1.21 g(99%) of the title compound.

¹NMR

C. 4-(4-morpholinyl)benzyl Alcohol

A solution of 4-(4-morpholinyl)benzoic acid (1.00 g, 4.83 mmol) and4-methylmorpholine (0.53 mL, 4.8 mmol) in tetrahydrofuran (25 mL) at−10° C. was treated with ethyl chloroformate (0.46 mL, 4.8 mmol). After0.25 h, the mixture was treated with sodium borohydride (550 mg, 14.5mmol) followed by MeOH (50 mL) slowly. The mixture was then treated with5% HOAC in water and the mixture was concentrated . The residue waspurified by chromatography (SiO₂, EtOAc:hexanes) yielding 164 mg (18%)of the title compound.

¹NMR, IR

FD-MS, m/e 193 (m)

Analysis for C₁₁H₁₅NO₂:

Calcd: C, 68.37; H, 7.82; N, 7.25;

Found: C, 68.46; H, 7.95; N, 7.21.

D. N¹-(4-methoxybenzoyl)-N²-[4-(4-morpholinyl)benzyl]-1,2-benzenediamine

4-(4-Morpholinyl)benzyl alcohol (150 mg, 0.78 mmol) was added to asolution of phosgene in toluene (1.93 M, 1.2 mL). After 4 h, the mixturewas treated with a solution of N¹-(4-methoxybenzoyl)-1,2-benzenediamine(188 mg, 0.78 mmol) and pyridine (2 mL) in methylene chloride (3 mL).After 16 h, the mixture was concentrated and the residue was dissolvedin EtOAc. The organic layer was washed with water (4×), brine (1×),dried with potassium carbonate, and concentrated. The residue waspurified by chromatography (SiO₂, 5 to 15% EtOAc in CH₂Cl₂), yielding 45mg (14%) of the title compound.

¹NMR, IR

FD-MS, m/e 417 (m)

Analysis for C₂₅H₂₇N₃O₃:

Calcd: C, 71.92; H, 6.52; N, 10.06;

Found: C, 72.12; H, 6.63; N, 10.11.

EXAMPLE 2 Preparation ofN¹-(4-Methoxybenzoyl)-N²-[1-(4-pyridyl)-piperidin-4-ylmethyl]-1,2-benzenediamine.

A. 1-(4-pyridyl)piperidine-4-methylamine

1-(4-Pyridyl)piperidine-4-methanol was prepared using a proceduresimilar to the following: A solution of methylN-(4-pyridyl)isonipecotate (600 mg, 2.72 mmol) in tetrahydrofuran wasadded to a solution of lithium aluminum hydride (100 mg) intetrahydrofuran (14 mL) cooled to 0° C. Upon consumption of the startingmaterial (0.5-2 h). the mixture was treated with water (0.10 mL), 15%aqueous sodium hydroxide (0.10 mL), and water (0.30 mL). After 0.25 h,the mixture was sonicated for 0.25 h, then poured into a mixture ofethyl acetate, water, sodium tartrate, and potassium tartrate. Theaqueous layer was extracted twice with ethyl acetate and the combinedextracts were dried (magnesium sulfate); filtered, and concentrated invacuo to yield 357 mg (68%) of 1-(4-pyridyl)piperidine-4-methanol, whichwas used without further purification.

¹H-NMR

A solution of 1-(4-pyridyl)piperidine-4-methanol (5.87 g, 30.6: mmol),phthalimide (4.59 g, 31.2 mmol), and triphenylphosphine (8.10 g, 30.9mmol) in 125 mL of THF at −5° C. was treated with a solution of diethylazodicarboxylate (5.38 g, 30.9 mmol) in THF (40 mL). After 16 h, themixture was poured into EtOAc and 1 N HCl. The aqueous layer was washedwith EtOAc (2×), pH adjusted to 12 by addition of 5 N NaOH, and washedwith EtOAc (3×). The combined organic extracts were dried (K₂CO₃) andconcentrated, yielding 8.45 g (86%). The crude material (5.47 g, 17.0mmol) was then treated with hydrazine hydrate (3.5 mL, 60.0 mmol) inEtOH (50 mL). The mixture was heated at 75° C. for 5 h, cooled, dilutedwith CH₂Cl₂ (100 mL), and cooled to 0° C. The solid was removed byfiltration; and the filtrate was concentrated, yielding 3.32 g of thetitle compound which was used without further purification.

¹H-NMR, IR

FD-MS, m/e 191 (m)

B. 2-nitro-N-[1-(4-pyridyl)piperidin-4-ylmethyl]aniline

A solution of 2-fluoronitrobenzene (0.13 mL, 1.3 mmol) and1-(4-pyridyl)piperidine-4-methylamine (242 mg, 1.27 mmol) in DMF (5 mL)was treated with potassium carbonate (175 mg, 1.3 mmol). After 16 h, themixture was diluted with EtOAc, the organic layer was washed with water(3×), brine, dried with K₂CO₃, and concentrated. The residue wasdissolved in 5% HOAc in MeOH and loaded onto an SCX ion exchange column.Elution with MeOH followed by 2 M NH₃ in MeOH yielded 200 mg of thetitle compound; which was used without further purification.

¹NMR

C. N¹-[1-(4-pyridyl)piperidin-4-ylmethyl]-1,2-benzene-diamine

A mixture of 2-nitro-N-[1-(4-pyridyl)piperidin-4-yl-methyl]aniline (235mg, 0.75 mmol) and 10% palladium on carbon (200 mg) in ethanol (4 mL)was placed under an atmosphere of hydrogen gas. After 1 h, the mixturewas filtered through diatomaceous earth. The filtrate was concentratedyielding 212 mg of the title compound, which was used without furtherpurification.

¹NMR

D.N¹-(4-methoxybenzoyl)-N²-[1-(4-pyridyl)piperidin-4-yl-methyl]-1,2-benzendiamine

A solution of N¹-[1-(4-pyridyl)piperidin-4-ylmethyl]-1,2-benzenediamine(212 mg, 0.75 mmol) and pyridine (4 mL) in chloroform (2 mL) at 0° C.was treated with a solution of 4-methoxybenzoyl chloride (128 mg, 0.75mmol) in chloroform. The mixture was allowed to warm to room temperatureand stir for 17 h. The mixture was concentrated and the residue wasdissolved in EtOAc. The organic layer was washed with 1 N NaOH (2×),water (3×), brine (1×), dried (K₂CO₃), and concentrated. The residue waspurified by RPHPLC, yielding 52 mg (17%) of the title compound as ahydrochloride salt.

¹NMR

FD-MS, m/e 417 (m+1)

EXAMPLE 3 Preparation ofN¹-(4-Chlorobenzoyl)-N²-[1-(4-pyridyl)-piperidin-4-ylmethyl]-1,2-benzendiamine.

Using a similar procedure to that described in Example 2, Part D,N¹-[1-(4-pyridyl)piperidin-4-ylmethyl)-1,2-benzenediamine (167 mg, 0.59mmol) and 4-chlorobenzoyl chloride (0.075 mL, 0.59 mmol) yielded 137 mg(55%) of the title compound.

¹NMR, IR

IS-MS, m/e 419 (p-1)

EXAMPLE 4 Preparation ofN³-(4-Methoxybenzoyl)-N²-[1-(4-pyridyl)-piperidin-4-ylmethyl]-2,3-pyridinediamine.

A. 3-nitro-N-[1-(4-pyridyl)piperidin-4-ylmethyl]pyridine-2-amine

A solution of 2-chloro-3-nitropyridine (290 mg, 1.83 mmol),triethylamine (0.26 mL) and 1-(4-pyridyl)-piperidine-4-methylamine (350mg, 1.83 mmol) in EtOH (10 mL) was heated at reflux. After 17 h, themixture was concentrated and the residue was purified by chromatography(SiO₂, 2 to 4% (2 N NH₃ in methanol) in chloroform) yielding 340 mg(60%) of the title compound.

¹NMR, IR

IS-MS, m/e 314 (m+1)

Analysis for C₂₅H₂₇N₃O₃CH₃OH:

Calcd: C, 59.12; H, 6.71; N, 20.28;

Found: C, 59.00; H, 6.87; N, 20.45.

B. N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridine-diamine

Using a similar procedure to that described in Example 2, Part C,3-nitro-N-[1-(4-pyridyl)piperidin-4-ylmethyl]pyridine-2-amine (340 mg,1.09 mmol) and 10% palladium on carbon (200 mg) yielded 300 mg of thetitle compound, which was used without further purification.

¹NMR

C.N³-(4-methoxybenzoyl)-N²-[1-(4-pyridyl)piperidin-4-yl-methyl]-2,3-pyridinediamine

Using a similar procedure to that described in Example 2, Part D,N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridinediamine (300 mg, 1.06mmol) and 4-methoxybenzoyl chloride (200 mg, 1.17 mmol) yielded 67 mg(15%) of the title compound.

¹NMR, IR

IS-MS, m/e 418 (m+1)

EXAMPLE 5 Preparation ofN³-(4-Chlorobenzoyl)-N²-[1-(4-pyridyl)-piperidin-4-ylmethyl]-2,3-pyridinediamine.

Using a similar procedure to that described in Example 2, Part D,N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-1,2-pyridinediamine (300 mg, 1.06mmol) and 4-chlorobenzoyl chloride (200 mg, 1.17 mmol) yielded 118 mg(15%) of the title compound as a hydrochloride salt.

¹NMR, IR

IS-MS, m/e 422 (m+1)

Analysis for C₂₃H₂₄ClN₅O.0.5H₂O.2HCl:

Calcd: C, 54.83; H, 5.40; N, 13.90;

Found: C, 54.90: H, 5.59; N, 13.50.

EXAMPLE 6 Preparation ofN³-(3-Fluoro-4-methoxybenzoyl)-N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridinediamine

Using a similar procedure to that described in Example 2, Part D,N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridinediamine (300 mg, 1.06mmol) and 3-fluoro-4-methoxybenzoyl chloride (200 mg, 1.17 mmol) yielded118 mg (15%) of the title compound as a hydrochloride salt.

¹NMR, IR

IS-MS, m/e 436 (m+1)

Analysis for C₂₄H₂₆FN₅O₂.2HCl:

Calcd: C, 56.70; H, 5.55; N, 13.77;

Found: C, 56.55; H, 5.46; N, 13.68.

EXAMPLE 7 Preparation ofN³-(5-Chlorothiophen-2-ylcarbonyl)-N²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridinediamine

A solution of 5-chlorothiophene-2-carboxylic acid (120 mg, 0.72 mmol) inmethylene chloride and DMF (0.005 mL) was treated with oxalyl chloride(0.105 mL, 1.20 mmol). After 0.25 h, the mixture was concentrated andthe residue was dissolved in chloroform. This solution was addeddropwine to a solution ofN²-[1-(4-pyridyl)piperidin-4-ylmethyl]-2,3-pyridinediamine (170 mg, 0.6mmol) in pyridine and chloroform. The reaction mixture was then purifiedusing a procedure similar to that described in Example 2, Part D,yielding 160 mg (58%) of the title compound as a hydrochloride salt.

¹NMR, IR

IS-MS, m/e 428 (m+1)

Analysis for C₂₁H₂₂ClN₅OS.2HCl.0.5H₂O:

Calcd: C, 49.48; H, 4.94; N, 13.74;

Found: C, 49.40; H, 4.48; N, 13.30.

EXAMPLE 8 Preparation ofN-(4-Methoxyphenyl)-2-[1-(4-pyridyl)-piperidin-4-ylmethyl]aminopyridine-3-carboxamide

A. 2-chloro-N-(4-methoxyphenyl)pyridine-3-carboxamide

2-Chloronicotinyl chloride hydrochloride (2.94 g, 16.5 mmol) was addedin portions to a solution of pyridine (4.0 mL) and 4-anisidine (2 g,16.2 mmol) in chloroform. After 0.5 h, the mixture was poured into EtOAcand 1 N NaOH. The organic layer was washed with 1 N NaOH (1×), water(1×), dried over potassium carbonate, and concentrated. The residue waspurified by recrystallization (StOAc:hexanes) yielding 2.56 g (60%) ofthe title compound.

¹NMR, IR

FD-MS, m/e 262 (m)

Analysis for C₁₃H₁₁ClN₂O₂:

Calcd: C, 59.44; H, 4.22; N, 10.66;

Found: C, 59.64; H? 4.45; N, 10.51.

B.N-(4-methoxyphenyl)-2-[1-(4-pyridyl)piperidin-4-yl-methyl]aminopyridine-3-carboxamide

A pressure tube (Aldrich) was charged with2-chloro-N-(4-methoxyphenyl)pyridine-3-carboxamide (139 mg, 0.524 mmol),1-(4-pyridyl)piperidine-4-methylamine (100 mg, 0.524 mmol),triethylamine (0.22 mL), and ethanol (3 mL). The mixture was placed in a110° C. bath for 5 days. The mixture was concentrated and the residuepurified by RPHPLC, yielding 52 mg (24%) of the title compound as ahydrochloride salt.

¹NMR, IR

IS-MS, m/e 418 (m+1)

Analysis for C₂₅H₂₆N₅O₂.2HCl:

Calcd: C, 58.78; H, 5.96; N, 14.28;

Found: C, 58.74; H, 5.90; N, 13.91.

EXAMPLE 9 Preparation ofN-(4-Chlorophenyl)-2-[1-(4-pyridyl)piperidin-4-ylmethyl]aminopyridine-3-carboxamide

A. 2-chloro-N-(4-chlorophenyl)pyridine-3-carboxamide

Using a similar procedure to that described in Example 8, Part A,2-chloronicotinyl chloride hydrochloride (500 mg, 2.84 mmol) and4-chloroaniline (432 mg, 3.41 mmol) yielded 800 mg of the titlecompound.

¹NMR

B.N-(4-chlorophenyl)-2-[1-(4-pyridyl)piperidin-4-ylmethyl]aminopyridine-3-carboxamide

Using a similar procedure to that described in Example 8, Part B,2-chloro-N-(4-chlorophenyl)pyridine-3-carboxamide (125 mg, 0.47 mmol),1-(4-pyridyl)piperidin-4-ylmethylamine (90 mg, 0.47 mmol), andtriethylamine (0.07 mL) yielded 40 mg (24%) of the title compound.

¹NMR, IR

IS-MS, m/e 422 (p+)

Analysis for C₂₃H₂₄ClN₅O:

Calcd: C, 65.47; H, 5.73; N, 16.60;

Found: C, 65.26; H, 5.77; N, 16.36.

EXAMPLE 10 Preparation ofN³-(4-Methoxybenzoyl)-N⁴-[1-(4-pyridyl)-piperidin-4-ylmethyl]-3,4-pyridinediamine

A. 3-nitro-N-[1-(4-pyridyl)piperidin-4-ylmethyl]pyridine-4-amine

Using a similar procedure to that described in Example 4, Part A,4-methoxy-3-nitropyridine (250 mg, 1.62 mmol), and1-(4-pyridyl)piperidin-4-ylmethylamine (310 mg, 1.62 mmol) yielded 370mg (73%) of the title compound.

¹NMR

B. N⁴-[1-(4-pyridyl)piperidin-4-ylmethyl]-3,4-pyridine-diamine

Using a similar procedure to that described in Example 2, Part C,3-nitro-N-[1-(4-pyridyl)piperidin-4-ylmethyl]pyridine-4-amine (370 mg)yielded 110 mg (40%) of the title compound; which was purified by flashchromatography (SiO₂, 5 to 10% (2N NH₃ in MeOH) in chloroform).

¹NMR

C.N³-(4-methoxybenzoyl)-N⁴-[1-(4-pyridyl)piperidin-4-yl-methyl]-3,4-pyridinediamine

Using a similar procedure to that described in Example 2, Part D,N⁴-[1-(4-pyridyl)piperidin-4-ylmethyl]-3,4-pyridinediamine (110 mg,0.388 mmol) and 4-methoxybenzoyl chloride (0.66 mL, 0.388 mmol) yielded10 mg (6%) of the title compound as a hydrochloride salt.

¹NMR

IS-MS, m/e 418 (m+1)

EXAMPLE 11 Preparation ofN-(4-Chlorophenyl)-3-[1-(4-pyridyl)piperidin-4-ylmethyl]aminopyridine-4-carboxamide

A. 3-Chloro-N-(4-chlorophenyl)pyridine-4-carboxamide

A solution of 3-chloropyridine (1.00 mL, 10.5 mmol) in THF at −78° C.was treated dropwise with a solution of lithium diisopropylamide in THF[freshly prepared by addition of butyllithium (7.21 mL, 11.5 mmol) todiisopropylamine (11.5 mmol)]. After 0.25 h, the mixture was treatedwith carbon dioxide (g) and slowly warmed to ambient temperature. Themixture was concentrated, partitioned between EtOAc and water, and theaqueous layer was washed with EtOAc (2×). The pH of the aqueous layerwas adjusted (˜3) by addition of 1 N HCl and then washed with EtOAc(3×). The combined extracts were dried with magnesium sulfate andconcentrated. The residue was recrystallized from EtOAC yielding 200 mg(12%) of 3-chloroisonicotinic acid.

A solution of the acid (200 mg) in methylene chloride (6 mL) anddimethyl formamide (0.01 mL) was treated with oxalyl chloride (0.22 mL,2.55 mmol). After 0.25 h, the mixture was concentrated, the residuedissolved in methylene chloride (6 mL) and then added dropwise to asolution of 4-chloroaniline (323 mg, 2.55 mmol) in pyridine (4 mL).After 1 h, the mixture was concentrated, the residue partitioned betweenEtOAc and water, the organic layer was washed with 1 N NaOH, brine, anddried with sodium sulfate; then concentrated. The residue was purifiedby column chromatography (SiO₂, 2:3 EtOAc:hexanes) yielding 130 mg (38%)of the title compound.

¹NMR

IS-MS, m/e 265 (m−1)

B.N-(4-chlorophenyl)-2-[1-(4-pyridyl)piperidin-4-yl-methyl]aminopyridine-3-carboxamide

A mixture of 3-chloro-N-(4-chlorophenyl)pyridine-4-carboxamide (130 mg,0.49 mmol), 1-(4-pyridyl)piperidine-4-methylamine (187 mg, 0.98 mmol),and copper (1) bromide (70 mg) in dimethylformamide (1 mL) was heated at110° C. After 18 h, the mixture was diluted with MeOH, filtered, andconcentrated. The residue was treated with 6:1 chloroform:water (10 mL)followed by MeOH until a homogenoeus solution resulted. The solution wastreated with hydrogen sulfide (g), heated at reflux for 0.1 h, andfiltered through diatomaceous earth. The filtrate was concentrated andthe residue taken up in water, 1 N NaOH, and EtOAC. The aqueous layerwas washed with EtOAc (3×), the combined extracts were dried (sodiumsulfate), filtered and concentrated. The residue was purified by RPHPLCyielding 29 mg (13%) of the title compound as a hydrochloride salt.

¹NMR

IS-MS, m/e 420 (m−1)

Preparation ofN-(6-Indolyl)-2-[1-(4-pyridyl)piperidin-4-ylmethyl]aminopyridine-3-carboxamide

A. Ammonium2-[1-(4-pyridyl)piperidin-4-ylmethyl]amino-pyridine-3-carboxylate

A mixture of 2-chloronicotinic acid (10.74 g, 67.5 mmol),1-(4-pyridyl)piperidine-4-methylamine (8.60 g, 45.0 mmol), and potassiumcarbonate 15.5 g, 112.6 mmol) in dimethylformide (90 mL) was heated atreflux. After 16 h, the mixture was diluted with methanol, filtered, andconcentrated. The residue was dissolved in methanol, acidified with 1 NHCl in ether, heated at reflux for 0.25 h, cooled, and the solid removedby filtration. The filtrate was then treated with 2 M NH₃ in methanoluntil slightly basic, triturated with THF and the resulting solidcollected by filtration yielding 10.75 g of the title compound which wasused without further purification.

¹NMR

IS-MS, m/e 313 (m+1)

B.N-(6-indolyl)-2-[1-(4-pyridyl)piperidin-4-ylmethyl]-aminopyridine-3-carboxamide

A solution of ammonium2-[1-(4-pyridyl)piperidin-4-yl-methyl]aminopyridine-3-carboxylate (3.0g, 9.12 mmol) in dioxane (45 mL) was treated with phosgene (1.9 M intoluene, 9.50 mL, 18.2 mmol) and the resulting mixture was heated atreflux. After 2 h, the mixture was concentrated yielding thecorresponding 4-azaisatoic anhydride which was used without furtherpurification. A solution of the crude anhydride (450 mg, 0.972 mmol) inTHF (5 mL) at −78° C. was treated with the magnesium salt of6-amino-1-tert-butoxy-carbonylindole [3.89 mmol; freshly prepared byaddition of methyl magnesium bromide (3.0 M in THF, 1.30 mL, 3.89 mmol)to 6-amino-1-tert-butoxycarbonylindole (900 mg, 3.89 mmol) in THF (10mL) at −78° C.]. After 17 h, the mixture was treated with a saturatedaqueous solution of ammonium chloride, diluted with water, andpartitioned between EtOAC. The aqueous layer was washed with EtOAc (3×)and the combined extracts were washed with water (1×), dried with sodiumsulfate, and concentrated. The residue was purified by columnchromatography (SiO₂, 6% (2M NH₃ in MeOH) in chloroform) yielding 140 mg(28%) of the coupled product. The coupled product was melted, cooled,and purified by RPHPLC yielding 67 mg (50%) of the title compound.

¹NMR, IR

IS-MS, m/e 427 (m+1)

Analysis for C₂₅H₂₆N₅O.HCl.H₂O:

Calcd: C, 62.43; H, 6.08; N, 17.47;

Found: C, 62.25; H, 5.81; N, 17.51.

EXAMPLE 13 Preparation ofN-(4-Chlorophenyl)-2-[1-(4-pyridyl)-piperidin-4-ylmethyl]aminopyridine-3-methylamine

A solution ofN-(4-chlorophenyl)-2-[1-(4-pyridyl)-piperidin-4-ylmethyl]aminopyridine-3-carboxamide(25 mg, 0.059 mmol) in THF (1 mL) was treated with lithium aluminumhydride (1.0 M in THF, 0.21 mL, 0.21 mmol). The mixture was heated at65° C. for 4 days, cooled, diluted with methanol (5 mL), and loaded ontoan SCX ion exchange resin. The resin was eluted with methanol followedby 2 N NH₃ in methanol and fractions containing the desired materialwere concentrated. The residue was triturated with EtOAc, yielding 15 mg(62%) of the title compound.

¹NMR

IS-MS, m/e 409 (M+1)

EXAMPLE 14 Preparation of5-Chloro-2-(1-isopropylpiperidin-4-ylmethyl-amino)-N-(2-pyridyl)benzamide

A. 1-Boc-piperidine-4-methanol

To a solution of Boc-isonipecotic acid (40 g, 0.17 mol) andN-methylmorpholine (19 mL, 0.17 mol) in tetrahydrofuran (900 mL)stirring at −10° C., ethyl chloroformate was slowly added (17 mL, 0.17mol) via addition funnel. After 30 min, sodium borohydride was added(19.8 g, 0.5 mol) in one portion. The reaction mixture was stirred at−10° C. for 1 h; then it was slowly quenched with methanol. The solventwas removed in vacuo: the resulting residue was diluted with 10% aqueousacetic acid and partitioned between ethyl acetate and water. The aqueouslayer was separated and extracted with additional ethyl acetate (2×500mL). The combined organic extracts were dried with magnesium sulfate,filtered, and concentrated in vacuo to a solid residue which waschromatographed on silica gel. Elution with ethyl acetate-hexanes (1:9to 1:1) provided the title compound (33.8 g, 90%) as a white solid.

¹NMR

B. 1-Boc-piperidine-4-carboxamide

A solution containing oxalyl chloride (8 mL, 87 mmol), indichloromethane (80 mL) at −78° C. was treated with dimethyl sulfoxide(12 mL, 0.17 mol). After stirring for 15 minutes,1-Boc-piperidine-4-methanol from above (3.7 g, 17 mmol) was added as asolution in dichloromethane (35 mL) via cannulation. The solution wasthen stirred at −78° C. for 1 h, after which triethylamine (36 mL, 0.26mol) was added dropwise to the cold solution. The reaction mixture wasallowed to warm to room temperature, upon which a thick white slurryformed. The mixture was poured into a saturated aqueous ammoniumchloride solution (200 mL); then the organic layer was separated and theaqueous layer was extracted with dichloromethane (75 mL). The organiclayers were combined and washed with brine (75 mL), then dried withmagnesium sulfate. The organic phase was filtered and concentrated invacuo, and the residue was redissolved in a 1:1 mixture of ethyl acetateand hexane and filtered through a Florisil plug (100-200 mesh). Theresulting filtrate was concentrated in vacuo to give 3.9 g (100%) of thetitle aldehyde as a yellow oil, which was used in the next step withoutfurther purification.

¹NMR

C. 2-amino-5-chloro-N-(2-pyridyl)benzamide

To a solution of 5-chloro-2-nitrobenzoic acid (15 g, 74 mmol) indichloromethane (300 mL) containing a few drops ofN,N-dimethylformamide, oxalyl chloride was slowly added (7.9 mL, 89mmol). After 2 h at room temperature, the solvent was removed in vacuoand the residue was redissolved in dichloromethane (300 mL). Theresulting solution was then treated with pyridine (18 mL, 0.2 mol),followed by 2-aminopyridine (7 g, 74 mmol). After 16 h at roomtemperature, the reaction mixture was concentrated in vacuo to a residuethat was partitioned between ethyl acetate and water. The organic phasewas separated and washed sequentially with 1 M aqueous citric acid,brine, saturated aqueous sodium bicarbonate, and brine. The organiclayer was dried with magnesium sulfate, filtered and concentrated to asmall volume. The concentrated solution was then diluted with diethylether, which caused the formation of a white precipitate. Sonicationfollowed by filtration provided a white solid (8.6 g, 42%), which wasdissolved in ethyl acetate-tetrahydrofuran (1:1) and submitted tohydrogen pressure (4.1 bar) in the presence of catalytic Raney-Nickel(0.8 g) for 16 h at room temperature. The reaction mixture was filteredthrough diatomaceous earth, and the filtrate was concentrated in vacuoto a solid residue which was purified via silica gel chromatography.Elution with ethyl acetate-hexanes (3:7) provided the title compound asa light-brown solid (4.4 g), which was used directly in the next stepwithout further purification.

¹NMR

FD-MS, m/e 248.0 (m).

D.2-(1-Boc-piperidin-4-ylmethylidinylimino)-5-chloro-N-(2-pyridyl)benzamide

A solution containing 1-Boc-piperidine-4-carboxamide from above (3.7 g,17 mmol), 2-amino-5-chloro-N-(2-pyridyl)-benzamide from above (4.3 g, 17mmol), and pyridinium p-toluenesulfonate (0.4 g, 1.7 mmol) in benzene(250 mL) was heated at reflux for 24 h with azeotropic removal of water.The mixture was then allowed to cool to room temperature and the solventwas removed in vacuo. The residue was partitioned between ethyl acetate(300 mL) and water (150 mL). The organic phase was separated and washedagain with water (150 mL) and then brine (150 mL); then it was driedwith magnesium sulfate, filtered and concentrated in vacuo to give 6.2 g(79%) of the desired imine as an orange foam which was used directly inthe next step without further purification.

¹NMR

FD-MS, m/e 443.1 (m)

E. 2-(1-Boc-piperidin-4-ylmethylamino)-5-chloro-N-(2-pyridyl)benzamide

A solution containing2-(1-Boc-piperidin-4-yl-methylidinylimino)-5-chloro-N-(2-pyridyl)benzamidefrom above (6.1 g, 14 mmol) and borane-trimethylamine complex (3.0 g, 41mmol) in glacial acetic acid (100 mL) was heated at 70° C. for 24 h.After cooling to room temperature, the solvent was removed in vacuo; andthe residue was partitioned between dichloromethane (200 mL) and water(100 mL). The solution was treated with 2 N sodium hydroxide untilneutral; then the organic layer was separated and the aqueous layer waswashed again with dichloromethane (100 mL). The combined organicextracts were washed with brine (100 mL), then dried with magnesiumsulfate and filtered. The filtrate was concentrated in vacuo to give6.23 g (100%) of the title compound as an orange foam, which was useddirectly in the next step without further purification.

¹NMR

FD-MS, m/e 445.2 (m)

F. 5-chloro-2-(piperidin-4-ylmethylamino)-N-(2-pyridyl)-benzamide

A solution of2-(1-Boc-piperidin-4-ylmethylamino)-5-chloro-N-(2-pyridyl)benzamide (6.1g, 14 mmol) in trifluoroacetic acid (125 mL) was stirred at 70° C. for 2h, then at room temperature for 24 h. The solution was concentrated invacuo, and the residue was directly applied to a silica gel column.Elution with dichloromethane—2 M ammonia in methanol (9:1) afforded 4.2g (89%) of the pure title compound, as a yellow solid.

¹NMR

FD-MS, m/e 345.1 (m)

Analysis for C₁₈H₂₀ClFN₄O.0.57CH₂Cl₂:

Calcd: C, 56.71; H, 5.67; N, 14.25;

Found: C, 56.91; H, 5.61; N, 13.85.

G.5-chloro-2-(1-isopropylpiperidin-4-ylmethylamino)-N-(2-pyridyl)benzamide

A solution of5-chloro-2-(piperidin-4-ylmethylamino)-N-(2-pyridyl)benzamide from above(1.3 g, 3.7 mmol) in acetone (28 mL) and methanol-acetic acid (95:5) (12mL) was treated with sodium cyanoborohydride (1.0 g, 15.0 mmol). Gasevolution was observed; the reaction mixture was then stirred at roomtemperature for 7 h, after which it was concentrated in vacuo to aresidue that was purified via silica gel chromatography. Elution withdichloromethane—2 M ammonia in methanol (9:1) provided 0.5 g (34%) ofthe title compound as a yellow solid.

¹NMR

mp 118-120° C.

FD-MS, m/e 387.2 (m)

Analysis for C₂₁H₂₇ClN₄O.0.25CH₂Cl₂:

Calcd: C, 62.53; H, 6.79; N, 13.72;

Found: C, 62.96; H, 6.73; N, 13.92.

EXAMPLE 15 Preparation ofN-(5-Chlorophenyl)-2-(1-isopropylpiperidin-4-ylmethyl)aminopyridine-3-carboxamide

A. 1-isopropylpiperidine-4-carboxamide

A solution of 200 mL of DMF, containing 50.0 g of isonicotinamide and 60mL of 2-bromopropane, was refluxed 5.75 h. A white insoluble solidfiltered from this cool solution gave 64.9 g (65%) of1-isopropylpyridinium-4-carboxamide bromide. m/e=165, NMR. Catalyticreduction of this salt, with PtO₂ in MeOH, gave 65.2 g (98%) of1-isopropylpiperidine-4-carboxamide hydrobromide, m/e=171. An aqueoussolution of this salt was gasified, evaporated to dryness, and extractedwith EtOAc to give 39.7 g (90%) of 1-isopropylpiperidine-4-carboxamidefree base.

B. 1-isopropylpiperidine-4-methylamine

To a suspension of 10.0 g of LAH in 500 mL of dry THF, at roomtemperature, was added portionwise 39.7 g of1-isopropylpiperidine-4-carboxamide and the mixture was refluxed 18 h.The cooled reaction mixture was diluted with 150 mL THF and treateddropwise with 10 mL H₂O and 10 mL 5 N NaOH, respectively. The resultinggray mixture was refluxed 18 h, filtered and evaporated. The residuepartially dissolved in hexane to give 25.5 g of crude yellow liquid and6.9 g hexane insoluble starting carboxamide. HPLC purification on silicagel of the 25.5 g liquid, eluting with 20% MeOH-EtOAc gave1-isopropylpiperidine-4-methylamine (8.5 g, 28%).

¹NMR

MS, m/e 157.

C. 2-chloro-N-(4-chlorophenyl)pyridine-3-carboxamide

By methods substantially equivalent to those described in Example 8,Part A, 2-chloro-N-(4-chlorophenyl)pyridine-3-carboxamide was preparedfrom 2-chloronicotinyl chloride and 4-chloroaniline.

D.N-(5-chlorophenyl)-2-(1-isopropylpiperidin-4-yl-methyl)aminopyridine-3-carboxamidehydrochloride

A solution of 0.20 g 2-chloro-N-(4-chlorophenyl)-pyridine-3-carboxamidein 5 mL of pyridine was treated with 0.23 g of1-isopropylpiperidine-4-methylamine and the mixture was refluxed 46.5 h.The cooled mixture was treated with 0.4 mL of 2 N NaOH and evaporated todryness. The EtOAc extract was purified by radial chromatography (10%MeOH in CHCl₃, 1% NH₄OH) to give 0.26 g of free base. The HCl salt wasisolated as an amorphous foam (0.24 g, 65%).

¹NMR

IS-MS, m/e 387 (M+1)

Analysis for C₂₁H₂₇ClN₄O.2HCl.1.75H₂O:

Calcd: C, 51.33; H, 6.67; N, 11.40;

Found: C, 50.85; H, 6.25; N, 11.22.

What is claimed is:
 1. A compound of formula I

(or a pharmaceutically acceptable salt thereof) wherein: A³, A⁴, A⁵ andA⁶, together with the two carbons to which they are attached, complete asubstituted benzene in which A³ is CR³, A⁴ is CR⁴, A⁵ is CR⁵, and A⁶ isCR⁶; wherein R³ is hydrogen; one of R⁴ and R⁵ is hydrogen, methyl,fluoro, chloro, R^(f)O₂C—, or R^(g)NH—; the other of R⁴ and R⁵ ishydrogen; and R⁶ is hydrogen; in which R^(f) is hydrogen, (1-4C)alkyl orbenzyl; R^(g) is hydrogen, or R^(h)SO₂—; and R^(h) is (1-4C)alkyl ordimethylamino; L¹ is —NH—CO—, —CO—H— or —CH₂—NH— such that —L¹—Q¹ is—NH—CO—Q¹ —CO—NH—Q¹ or —CH₂—NH—Q¹; Q¹ is phenyl or 2-naphthyl in whichthe phenyl may bear one, two or three substituents at the 3-, 4- or5-position(s) independently selected from halo, cyano, carbamoyl,aminomethyl, methyl, methoxy, difluoromethoxy, hydroxymethyl, formyl,vinyl, amino, hydroxy and 3,4-methylenedioxy; and in addition the phenylmay bear a 2-chloro or 2-fluoro substituent; or —CO—Q¹ iscyclopentenylcarbonyl or cyclohexenyl-carbonyl; and R² is —NH—CH₂—Q² inwhich Q² is Q^(2A) wherein Q^(2A) (showing the —CH₂— to which it isattached) is

 in which R^(2A) is 4-pyridinyl (which is unsubstituted or bears asubstituent R^(v) at the 2- or 3-position) wherein R^(v) is methyl,hydroxymethyl, {(1-2C)alkoxy}carbonyl; cyano, carbamoyl, thiocarbamoyl,or N-hydroxyamidino.
 2. The compound of claim 1 wherein halo is fluoro,chioro, bromo or iodo; (1-2C)alkyl is methyl or ethyl; and (1-4C)alkylis methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl.
 3. Thecompound as claimed in claim 2 wherein Q¹ is 4-chlorophenyl,4-methoxyphenyl, or 3-fluoro-4-methoxyphenyl.
 4. The compound as claimedin claim 2 wherein R² is [1-(4-pyridinyl)piperin-4-yl-methyl]amino. 5.The compound as claimed in any of claims 1-4 wherein each of R³-R⁶ ishydrogen, or each of R³, R⁴ and R⁶ is hydrogen and R⁵ is chloro.
 6. Thecompound as claimed in any one of claims 1-4 wherein —L¹—Q¹ is—CO—NH—Q¹.
 7. The pharmaceutically acceptable salt of a compound offormula I as claimed in claim 1 which is an acid-addition salt made froma basic compound of formula I and an acid which provides apharmaceutically acceptable anion or a salt which is made from an acidiccompound of formula I and a base which provides a pharmaceuticallyacceptable cation.
 8. A pharmaceutical formulation comprising inassociation with a pharmaceutically acceptable carrier, diluent orexcipient, a novel compound of formula I (or a pharmaceuticallyacceptable salt thereof) as provided in claim
 1. 9. A process forpreparing a compound of formula I (or a pharmaceutically acceptable saltthereof) as provided in claim 1 which is selected from (A) for acompound of formula I in which —L¹—Q¹ is —NH—CO—Q¹, acylating an amineof formula II,

 using a corresponding acid of formula HO—CO—Q¹, or an activatedderivative thereof; (B) for a compound of formula I in which —L¹—Q¹ is—CO—NH—Q¹, substituting the group y^(a) of a compound of formula III

 in which y^(a) is a conventional leaving group for nucleophilicaromatic substitution with an amine of formula NH₂—CH₂—Q²; (C) for acompound of formula I in which —L¹—Q¹ is —CO—NH—Q¹, acylating an amineof formula H₂N—Q¹, or a deprotonated derivative thereof, using an acidof formula IV, or an activated derivative thereof;

(D) alkylating an amine of formula V

 directly, using a compound of formula Y—CH₂—Q², or indirectly byreductive alkylation using an aldehyde of formula Q²—CHO; (E) for acompound of formula I in which —L¹—Q¹ is —CH₂—NH—Q¹, reducing acorresponding compound of formula I in which —L¹—Q¹ is —CO—NH—Q¹; (H)for a compound of formula I in which R^(2A) is 4-pyridinyl (which isunsubstituted or bears a substituent R^(v) at the 2- or 3-position),substituting the amino nitrogen of a corresponding compound of formula Iin which R^(2A) is hydrogen using a corresponding pyridine reagentbearing a leaving group Y at the 4-position; (I) for a compound offormula I in which R^(2A) is 4-pyridinyl in which R^(v) isalkoxycarbonyl, esterifying a corresponding compound of formula I inwhich R^(v) is carboxy; (J) for a compound of formula I in which R^(2A)is 4-pyridinyl in which R^(v) is hydroxymethyl, reducing the ester of acorresponding compound of formula I in which R^(v) is alkoxycarbonyl;(K) for a compound of formula I in which R^(2A) is 4-pyridinyl in whichR^(v) is carbamoyl, amidating the ester of a corresponding compound offormula I in which R^(v) is alkoxycarbonyl; (L) for a compound offormula I in which R^(2A) is 4-pyridinyl in which R^(v) isthiocarbamoyl, adding H₂S to the nitrile of a corresponding compound offormula I in which R^(v) is cyano; (M) for a compound of formula I inwhich R^(2A) is 4-pyridinyl in which R^(v) is N-hydroxyamidino, addingH₂NOH to the nitrile of a corresponding compound of formula I in whichR^(v) is cyano; (N) for a compound of formula I in which R^(2A) is4-pyridinyl in which R^(v) is carboxy, decomposing the ester of acorresponding compound of formula I in which R^(v) is alkoxycarbonyl;(S) for a compound of formula I in which R⁴ or R⁵ is amino, reducing thenitro group of a compound corresponding to a compound of formula I butin which R⁴ or R⁵ is nitro; and (T) for a compound of formula I in whichR⁴ or R⁵ is R^(g)NH— and R^(g) is R^(h)SO₂—, substituting the aminogroup of a corresponding compound of formula I in which R⁴ or R⁵ isamino using an activated derivative of the sulfonic acid R^(h)SO₂—OH;whereafter, for any of the above procedures, when a functional group isprotected using a protecting group, removing the protecting group;whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of formula I is required, it is obtainedby reacting the basic form of a basic compound of formula I with an acidaffording a physiologically acceptable counterion or the acidic form ofan acidic compound, of formula I with a base affording a physiologicallyacceptable counterion or by any other conventional procedure; andwherein, unless otherwise specified, A³-A⁶, L¹, Q¹ and R² have any ofthe values defined in claim
 1. 10. A method of inhibiting factor Xacomprising administering to a mammal in need of treatment, a compound offormula I as provided in claim
 1. 11. The compound as claimed in claim 5wherein —L¹—Q¹ is —CO—NH—Q¹.